ABSTRACT
GABAergic interneurons play a critical role in tuning neural networks in the central nervous system, and their defects are associated with neuropsychiatric disorders. Currently, the mDlx enhancer is solely used for adeno-associated virus (AAV) vector-mediated transgene delivery into cortical interneurons. Here, we developed a new inhibitory neuron-specific promoter (designated as the mGAD65 promoter), with a length of 2.5 kb, from a mouse genome upstream of exon 1 of the Gad2 gene encoding glutamic acid decarboxylase (GAD) 65. Intravenous infusion of blood-brain barrier-penetrating AAV-PHP.B expressing an enhanced green fluorescent protein under the control of the mGAD65 promoter transduced the whole brain in an inhibitory neuron-specific manner. The specificity and efficiency of the mGAD65 promoter for GABAergic interneurons, which was assessed at the motor cortex, were almost identical to or slightly higher than those of the mDlx enhancer. Immunohistochemical analysis revealed that the mGAD65 promoter preferentially transduced parvalbumin (PV)-expressing interneurons. Notably, the mGAD65 promoter transduced chandelier cells more efficiently than the mDlx enhancer and robustly labeled their synaptic boutons, called the cartridge, targeting the axon initial segments of excitatory pyramidal neurons. To test the ability of the mGAD65 promoter to express a functional molecule, we virally expressed G-CaMP, a fluorescent Ca2+ indicator, in the motor cortex, and this enabled us to monitor spontaneous and drug-induced Ca2+ activity in GABAergic inhibitory neurons. These results suggest that the mGAD65 promoter is useful for AAV-mediated targeting and manipulation of GABAergic neurons with the dominance of cortical PV-expressing neurons, including chandelier cells.
Subject(s)
Brain/metabolism , Dependovirus/metabolism , GABAergic Neurons/metabolism , Plasmids/metabolism , Transduction, Genetic , Animals , Calcium/metabolism , Glutamate Decarboxylase/metabolism , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Injections, Intravenous , Interneurons/metabolism , Mice, Inbred C57BL , Motor Cortex/metabolism , Neurons/metabolism , Parvalbumins/metabolism , Promoter Regions, GeneticABSTRACT
Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.
Subject(s)
Genetic Therapy , Receptors, Vascular Endothelial Growth Factor/genetics , Receptors, Vascular Endothelial Growth Factor/therapeutic use , Retinal Neovascularization/genetics , Retinal Neovascularization/therapy , Animals , Cell Hypoxia , Dependovirus/metabolism , Disease Models, Animal , Female , Gene Transfer Techniques , HEK293 Cells , Humans , Intravitreal Injections , Protein Domains , Rats, Sprague-Dawley , Tetrahydrofolate Dehydrogenase/chemistry , Tetrahydrofolate Dehydrogenase/metabolism , Transgenes , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Mitochondrial dysfunction mediated loss of respiration, oxidative stress, and loss of cellular homeostasis contributes to the neuronal and axonal degenerations permanent loss of function in experimental autoimmune encephalomyelitis model (EAE) of multiple sclerosis (MS). To address the mitochondrial dysfunction mediated visual loss in EAE mice, self-complementary adeno-associated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene was intravitreally injected into the mice after the onset of visual defects. Visual function assessed by pattern electroretinogram (PERGs) showed progressive loss of function in EAE mice were improved significantly in NDI1 gene therapy-treated mice. Serial optical coherence tomography (OCT) revealed that progressive thinning of inner retinal layers in EAE mice was prevented upon NDI1 expression. The 45% optic nerve axonal and 33% retinal ganglion cell (RGC) loss contributed to the permanent loss of visual function in EAE mice were ameliorated by NDI1-mediated prevention of mitochondrial cristae dissolution and improved mitochondrial homeostasis. In conclusion, targeting the dysfunctional complex I using NDI1 gene can be an approach to address axonal and neuronal loss responsible for permanent disability in MS that is unaltered by current disease modifying drugs.
Subject(s)
Electron Transport Complex I/genetics , Electron Transport Complex I/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Encephalomyelitis, Autoimmune, Experimental/therapy , Genetic Therapy , Multiple Sclerosis/physiopathology , Multiple Sclerosis/therapy , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/therapeutic use , Vision, Ocular , Animals , Axons/pathology , Dependovirus/metabolism , Disease Models, Animal , Electroretinography , Encephalomyelitis, Autoimmune, Experimental/diagnostic imaging , Mice , Mitochondria/metabolism , Mitochondria/ultrastructure , Multiple Sclerosis/diagnostic imaging , Optic Nerve/pathology , Optic Nerve/ultrastructure , Retina/metabolism , Retina/pathology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , Saccharomyces cerevisiaeABSTRACT
OBJECTIVE: Restoring calcium sensor protein (S100A1) activity in failing hearts poses a promising therapeutic strategy. We hypothesize that cardiac overexpression of the S100A1 gene mediated by a double-stranded adeno-associated virus (scAAV) results in better functional and molecular improvements compared with the single-stranded virus (ssAAV). METHODS: Heart failure was induced by coronary artery ligation. Then, intramyocardial injections of saline, AAV9 empty capsid, scAAV9.S100A1, and ssAAV9.S100A1 were performed. Ten weeks postinfarction, all rats received cardiac evaluation; serum and tissue were collected for genetic analysis, cytokine profiling, and assessments of mitochondrial function and structure. RESULTS: Overexpression of AAV9.S100A1 improved systolic and diastolic function. Compared with control, ejection fraction was greater in treated groups (54.8% vs 32.3%, P < .05). Similarly, end-diastolic volume index was significantly less in the treated group than in control (1.14 vs 1.59 mL/cm2), whereas fractional shortening was greater in treated groups than control (26% vs 38%, P < .05). Interestingly, cardiac mechanics were significantly better when treated with double-stranded virus compared with single-stranded. Quantitative polymerase chain reaction demonstrated robust transfection of myocardium with the S100A1 gene, with more infection in the self-complimentary group compared with the single-stranded group (5.68 ± 0.44 vs 4.09 ± 0.25 log10 genome copies per 100 ng of DNA; P < .0001). Concentrations of the inflammatory cytokines were elevated in the ssAAV9/S100A1 group compared with the scAAV9/S100A1. Assessment of mitochondrial respiration and morphology demonstrated that injection of self-complementary vector saved both mitochondrial structure and function. CONCLUSIONS: Gene therapy of S100A1 can prevent pathologic postmyocardial infarction remodeling and decrease inflammatory response in ischemic heart failure.
Subject(s)
Calcium Signaling , Dependovirus/genetics , Genetic Therapy , Genetic Vectors , Heart Failure/therapy , Heart Ventricles/metabolism , Myocardial Infarction/therapy , S100 Proteins/genetics , Transfection , Ventricular Function, Left , Ventricular Remodeling , Animals , Cytokines/metabolism , Dependovirus/metabolism , Disease Models, Animal , Fibrosis , Heart Failure/genetics , Heart Failure/metabolism , Heart Failure/physiopathology , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Inflammation Mediators/metabolism , Lipid Peroxidation , Male , Mitochondria, Heart/metabolism , Mitochondria, Heart/pathology , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardial Infarction/physiopathology , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , S100 Proteins/biosynthesis , Stroke VolumeABSTRACT
Orexin (also known as hypocretin) neurons in the hypothalamus play an essential role in sleep-wake control, feeding, reward, and energy homeostasis. The likelihood of anesthesia and sleep sharing common pathways notwithstanding, it is important to understand the processes underlying emergence from anesthesia. In this study, we investigated the role of the orexin system in anesthesia emergence, by specifically activating orexin neurons utilizing the designer receptors exclusively activated by designer drugs (DREADD) chemogenetic approach. With injection of adeno-associated virus into the orexin-Cre transgenic mouse brain, we expressed the DREADD receptor hM3Dq specifically in orexin neurons and applied the hM3Dq ligand clozapine to activate orexin neurons. We monitored orexin neuronal activities by c-Fos staining and whole-cell patch-clamp recording and examined the consequence of orexin neuronal activation via EEG recording. Our results revealed that the orexin-DREADD mice with activated orexin neurons emerged from anesthesia with significantly shorter latency than the control mice. As an indication of reduced pain sensitivity, these orexin-DREADD mice took longer to respond to the 55 °C thermal stimuli in the hot plate test and exhibited significantly less frequent licking of the formalin-injected paw in the formalin test. Our study suggests that approaches to activate the orexin system can be beneficial in postoperative recovery.
Subject(s)
Anesthesia Recovery Period , Hypothalamus/metabolism , Neurons/metabolism , Orexin Receptors/genetics , Orexins/genetics , Pain/genetics , Anesthetics, Inhalation , Animals , Clozapine/pharmacology , Dependovirus/genetics , Dependovirus/metabolism , Electroencephalography , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Hot Temperature , Hypothalamus/drug effects , Hypothalamus/physiopathology , Isoflurane , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurons/drug effects , Neurons/pathology , Orexin Receptors/metabolism , Orexins/metabolism , Pain/physiopathology , Pain/prevention & control , Pain Measurement , Patch-Clamp Techniques , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , Serotonin Antagonists/pharmacology , Stereotaxic TechniquesABSTRACT
Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of motoneurons in the primary motor cortex (pMO) and in spinal cord. However, the pathogenic process involves multiple subnetworks in the brain and functional MRI studies demonstrate an increase in functional connectivity in areas connected to pMO despite the ongoing neurodegeneration. The extent and the structural basis of the motor subnetwork remodeling in experimentally tractable models remain unclear. We have developed a new retrograde AAV9 to quantitatively map the projections to pMO in the SOD1(G93A) ALS mouse model. We show an increase in the number of neurons projecting from somatosensory cortex to pMO at presymptomatic stages, followed by an increase in projections from thalamus, auditory cortex and contralateral MO (inputs from 20 other structures remains unchanged) as disease advances. The stage- and structure-dependent remodeling of projection to pMO in ALS may provide insights into the hyperconnectivity observed in ALS patients.
Subject(s)
Amyotrophic Lateral Sclerosis/physiopathology , Dependovirus/metabolism , Motor Cortex/physiopathology , Amyotrophic Lateral Sclerosis/pathology , Animals , Dendritic Spines/metabolism , Disease Models, Animal , Disease Progression , Female , Humans , Male , Mice , Motor Cortex/pathology , Mutant Proteins/metabolism , Nerve Net/pathology , Nerve Net/physiopathology , Protein Folding , Pyramidal Cells/metabolism , Pyramidal Cells/pathology , Superoxide Dismutase/metabolism , Thalamus/pathology , Thalamus/physiopathologyABSTRACT
Self-complementary adeno-associated viral vector 9 (scAAV9) has been confirmed to be an efficient AAV serotype for gene transfer to the central nervous system (CNS). Neurotrophic factors have been considered to be therapeutic targets for amyotrophic lateral sclerosis (ALS). In the present study, we intramuscularly injected scAAV9 encoding human insulin-like growth factor 1 (hIGF1) into an hSOD1G93A ALS mouse model. We observed that scAAV9-hIGF1 significantly reduced the loss of motor neurons of the anterior horn in the lumbar spinal cord and delayed muscle atrophy in ALS mice. Importantly, IGF1 significantly delayed disease onset and prolonged the life span of ALS mice. In addition, scAAV9-hIGF1 protected motor neurons from apoptosis through upregulation of D-amino acid oxidase (DAO), which controls the level of D-serine. Moreover, to further verify these results, we used CRISPR-Cas9 system to target the central nervous system knockdown of IGF1. This experiment supported the continued investigation of neurotrophic factor gene therapies targeting the central nervous system as a potential treatment for ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , D-Amino-Acid Oxidase/metabolism , Dependovirus/metabolism , Insulin-Like Growth Factor I/administration & dosage , Superoxide Dismutase/metabolism , Up-Regulation , Animals , Apoptosis , Disease Models, Animal , Disease Progression , Female , Gene Transfer Techniques , Humans , Injections, Intramuscular , Male , Mice, Transgenic , Motor Neurons/metabolism , Muscle, Skeletal/metabolism , Muscular Atrophy/pathology , Phenotype , RNA, Guide, Kinetoplastida/metabolism , Serine/metabolism , Survival Analysis , Transduction, GeneticABSTRACT
Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre.
Subject(s)
Chromatography, Affinity/methods , Protein Biosynthesis , Ribosomes/metabolism , Viruses/metabolism , Animals , Biomarkers/metabolism , Dependovirus/metabolism , Female , Gene Expression Regulation , Green Fluorescent Proteins/metabolism , Hypothalamic Hormones/metabolism , Hypothalamus/metabolism , Male , Melanins/metabolism , Mice , Neurons/metabolism , Pituitary Hormones/metabolism , Reproducibility of Results , SerotypingABSTRACT
GM2 gangliosidoses, including Tay-Sachs disease and Sandhoff disease, are lysosomal storage disorders caused by deficiencies in ß-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system (CNS) dysfunction. Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or ß-subunits at a 1:1 ratio. Here, a safety study was conducted in cynomolgus macaques (cm), modeling previous animal studies, with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and ß-subunits. Three doses (3.2 × 1012 vg [n = 3]; 3.2 × 1011 vg [n = 2]; or 1.1 × 1011 vg [n = 2]) were tested, with controls infused with vehicle (n = 1) or transgene empty AAVrh8 vector at the highest dose (n = 2). Most monkeys receiving AAVrh8-cmHexα/ß developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose dependent, with the highest-dose cohort producing symptoms within a month of infusion. One monkey in the lowest-dose cohort was behaviorally asymptomatic but had magnetic resonance imaging abnormalities in the thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/ß, showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus, and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome, and this study demonstrates the variations in safety profiles of AAVrh8-Hexα/ß intracranial injection among different species, despite encoding for self-proteins.
Subject(s)
Dependovirus/genetics , Dyskinesias/etiology , Gangliosidoses, GM2/therapy , Genetic Vectors/adverse effects , Necrosis/etiology , Neurons/metabolism , beta-N-Acetylhexosaminidases/genetics , Animals , Apathy , Dependovirus/metabolism , Disease Models, Animal , Dyskinesias/genetics , Dyskinesias/metabolism , Dyskinesias/pathology , Female , Gangliosidoses, GM2/genetics , Gangliosidoses, GM2/metabolism , Gangliosidoses, GM2/pathology , Gene Expression , Genetic Therapy/methods , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Gray Matter/metabolism , Gray Matter/pathology , Injections, Intraventricular , Macaca fascicularis , Male , Necrosis/genetics , Necrosis/metabolism , Necrosis/pathology , Neurons/pathology , Protein Subunits/adverse effects , Protein Subunits/genetics , Protein Subunits/metabolism , Thalamus/metabolism , Thalamus/pathology , Transgenes , White Matter/metabolism , White Matter/pathology , beta-N-Acetylhexosaminidases/adverse effects , beta-N-Acetylhexosaminidases/metabolismABSTRACT
The incidence of melanoma in the United States continues to rise, with metastatic lesions notoriously recalcitrant to therapy. There are limited effective treatment options available and a great need for more effective therapies that can be rapidly integrated in the clinic. In this study, we demonstrate that the combination of RGD-targeted adeno-associated virus phage (RGD-AAVP-TNF) with hypofractionated radiation therapy results in synergistic inhibition of primary syngeneic B16 melanoma in a C57 mouse model. Furthermore, this combination appeared to modify the tumor microenvironment, resulting in decreased Tregs in the draining LN and increased tumor-associated macrophages within the primary tumor. Finally, there appeared to be a reduction in metastatic potential and a prolongation of overall survival in the combined treatment group. These results indicate the use of targeted TNF gene therapy vector with radiation treatment could be a valuable treatment option for patients with metastatic melanoma.
Subject(s)
Dependovirus/genetics , Dependovirus/metabolism , Genetic Vectors/genetics , Melanoma/genetics , Melanoma/pathology , Oligopeptides/metabolism , Tumor Necrosis Factor-alpha/genetics , Animals , Cell Line, Tumor , Combined Modality Therapy , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genetic Vectors/adverse effects , Melanoma/metabolism , Melanoma/therapy , Melanoma, Experimental , Mice , Neoplasm Metastasis , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/therapy , Radiotherapy/methods , Radiotherapy, Image-Guided , Survival Analysis , Treatment Outcome , Tumor Burden/genetics , Tumor Burden/immunology , Tumor Burden/radiation effects , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
PURPOSE: We examined the neuroprotective effects of exogenous brain-derived neurotrophic factor (BDNF), which provides protection to retinal ganglion cells (RGCs) in rodents, in a model of transient intraocular pressure (IOP) elevation using a mutant (triple Y-F) self-complementary adeno-associated virus type 2 vector encoding BDNF (tm-scAAV2-BDNF). METHODS: The tm-scAAV2-BDNF or control vector encoding green fluorescent protein (GFP; tm-scAAV2-GFP) was intravitreally administered to rats, which were then divided into four groups: control, ischemia/reperfusion (I/R) injury only, I/R injury with tm-scAAV2-GFP, and tm-scAAV2-BDNF. I/R injury was then induced by transiently increasing IOP, after which the rats were euthanized to measure the inner retinal thickness and cell counts in the RGC layer. RESULTS: Intravitreous injection of tm-scAAV2-BDNF resulted in high levels of BDNF expression in the neural retina. Histological analysis showed that the inner retinal thickness and cell numbers in the RGC layer were preserved after transient IOP elevation in eyes treated with tm-scAAV2-BDNF but not in the other I/R groups. Significantly reduced glial fibrillary acidic protein (GFAP) immunostaining after I/R injury in the rats that received tm-scAAV2-BDNF indicated reduced retinal stress, and electroretinogram (ERG) analysis confirmed preservation of retinal function in the tm-scAAV2-BDNF group. CONCLUSIONS: These results demonstrate the feasibility and effectiveness of neuroprotective gene therapy using tm-scAAV2-BDNF to protect the inner retina from transiently high intraocular pressure. An in vivo gene therapeutic approach to the clinical management of retinal diseases in conditions such as glaucoma, retinal artery occlusion, hypertensive retinopathy, and diabetic retinopathy thus appears feasible.
Subject(s)
Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/therapeutic use , Dependovirus/metabolism , Intraocular Pressure , Mutation/genetics , Tyrosine/genetics , Animals , Cell Count , Disease Models, Animal , Electroretinography , Glial Fibrillary Acidic Protein/metabolism , Green Fluorescent Proteins/metabolism , Humans , Rats, Sprague-Dawley , Retina/injuries , Retina/pathology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , Transduction, GeneticSubject(s)
Biological Products/therapeutic use , Biological Therapy/methods , Heart Failure/therapy , Macular Degeneration/therapy , Parkinson Disease/therapy , Animals , Dependovirus/metabolism , Ear, Inner/drug effects , Gene Library , Genetic Vectors , Ghrelin/therapeutic use , Heart/drug effects , Humans , Ischemia/pathology , Myocytes, Cardiac/drug effects , Neurons/drug effects , Peptides/chemistry , Phenotype , Retina/drug effectsABSTRACT
In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.
Subject(s)
Calcium-Binding Proteins/metabolism , Calcium/metabolism , Dependovirus/metabolism , Gene Silencing , Genetic Vectors/metabolism , MicroRNAs/metabolism , Myocytes, Cardiac/metabolism , Animals , Blotting, Western , DNA, Complementary/genetics , Genes, Reporter , HEK293 Cells , Humans , Inflammation/pathology , Myocardium/metabolism , Proteomics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Wistar , Reproducibility of Results , Sarcoplasmic Reticulum , Transduction, GeneticABSTRACT
Considerable evidence has been published demonstrating the importance of lipoxygenase enzymes for vascular smooth muscle cell (VSMC) growth. The current study sets out to determine whether or not 12-lipoxygenase (12LO) is also important for human placental VSMC survival. Both a pharmacological and two 12LO antisense knockdown approaches were applied. The 12LO inhibitor baicalien induced a 2-2.5-fold increase in cell death, which appeared to result from apoptosis, as indicated by DNA fragmentation, activation of procaspase 3 to caspase 3 and cytochrome C release from the mitochondria to the cytosol. This apoptosis could be prevented by treatment with the 12LO product, 12 hydroxyeicosatetraenoic acid (12HETE). Human platelet-type 12LO-antisense knockdown, by either plasmid transfection or adeno-associated virus (AAV) infection also induced substantial VSMC death over controls, which could also be prevented by treatment with 12HETE, but not 5HETE. Hence, biochemical 12LO inhibition or 12LO-antisense knockdown in VSMC can induce programmed cell death. These observations suggest a previously unrecognized association between human VSMC survivability and 12LO.
Subject(s)
Arachidonate 12-Lipoxygenase/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/enzymology , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/pharmacology , Apoptosis , Arachidonate 12-Lipoxygenase/genetics , Biological Transport , Caspase 3/metabolism , Cell Survival , Cells, Cultured , Cytochromes c/metabolism , DNA, Complementary/genetics , DNA, Complementary/metabolism , Dependovirus/genetics , Dependovirus/metabolism , Flavanones/pharmacology , Gene Knockdown Techniques , Humans , Lipoxygenase Inhibitors/pharmacology , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Muscle, Smooth, Vascular/enzymology , Plasmids/genetics , Plasmids/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , TransfectionABSTRACT
We sought to determine a role for NPY overexpression in the dorsomedial hypothalamus (DMH) in obesity etiology using the rat model of adeno-associated virus (AAV)-mediated expression of NPY (AAVNPY) in the DMH. Rats received bilateral DMH injections of AAVNPY or control vector and were fed on regular chow. Five-week postviral injection, half the rats from each group were switched to access to a high-fat diet for another 11 weeks. We examined variables including body weight, food intake, energy efficiency, meal patterns, glucose tolerance, fat mass, plasma insulin, plasma leptin, and hypothalamic gene expression. Rats with DMH NPY overexpression had increased food intake and body weight and lowered metabolic efficiency. The hyperphagia was mediated through increased meal size during the dark. Although these rats had normal blood glucose, their plasma insulin levels were increased in both basal and glucose challenge conditions. While high-fat diet induced hyperphagia, obesity, and hyperinsulinemia, these effects were amplified in rats with DMH NPY overexpression. Arcuate Npy, agouti-related protein and proopiomelanocortin expression was appropriately regulated in response to positive energy balance. These results indicate that DMH NPY overexpression can cause hyperphagia and obesity and DMH NPY may have actions in glucose homeostasis.
Subject(s)
Hyperphagia/genetics , Hypothalamus/metabolism , Neuropeptide Y/genetics , Obesity/genetics , Agouti-Related Protein/genetics , Agouti-Related Protein/metabolism , Animals , Body Weight , Dependovirus/genetics , Dependovirus/metabolism , Diet, High-Fat , Disease Models, Animal , Energy Metabolism , Gene Expression Regulation , Glucose/metabolism , Glucose Tolerance Test , Homeostasis , Hyperphagia/pathology , Leptin/blood , Male , Neuropeptide Y/metabolism , Obesity/pathology , Pro-Opiomelanocortin/genetics , Pro-Opiomelanocortin/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The regulation of bone and fat homeostasis and its relationship to energy expenditure has recently been the focus of increased attention because of its potential relevance to osteoporosis, obesity, and diabetes. Although central effectors within the hypothalamus have been shown to contribute to the regulation of both energy balance and bone homeostasis, little is known of the underlying mechanisms, including the possible involvement of transcriptional factors within the hypothalamus. Transgenic mice overexpressing ΔFosB, a splice variant of the AP-1 transcription factor FosB with mixed agonist-antagonistic properties, have increased energy expenditure and bone mass. Because these mice express ΔFosB in bone, fat, and hypothalamus, we sought to determine 1) whether overexpression of ΔFosB within the hypothalamus was sufficient to regulate energy expenditure and whether it would also regulate bone mass, and 2) whether these effects were the result of antagonism to AP-1. Our results show that stereotactic injection of an adeno-associated virus vector to restrict overexpression of ΔFosB to the ventral hypothalamus of wild-type mice induced a profound increase in both energy expenditure and bone formation and bone mass. This effect was phenocopied, at an even stronger level, by overexpression of a dominant-negative DNJunD, a pure AP-1 antagonist. Taken together, these results suggest that downregulation of AP-1 activity in the hypothalamus profoundly increases energy expenditure and bone formation, leading to both a decrease in adipose mass and an increase in bone mass. These findings may have physiological implications because ΔFosB is expressed and regulated in the hypothalamus.
Subject(s)
Energy Metabolism/physiology , Hypothalamus/metabolism , Osteogenesis/physiology , Transcription Factor AP-1/metabolism , Animals , Body Weight/physiology , Dependovirus/metabolism , Genes, Dominant , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation/genetics , Organ Size , Protein Binding , Protein Isoforms/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Transcription Factor AP-1/antagonists & inhibitors , Transcription, GeneticABSTRACT
Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1α has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1α signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, we hypothesized that over-expression of PGC-1α in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1α. PGC-1α over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1α over-expressing muscle when compared to control. We also evaluated if the use a pharmacological PGC-1α pathway activator, resveratrol, could drive the same physiological changes. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1α pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle.
Subject(s)
Muscle Fibers, Fast-Twitch/pathology , Muscle Fibers, Slow-Twitch/pathology , Muscular Dystrophy, Animal/physiopathology , Recovery of Function/physiology , Trans-Activators/metabolism , Animals , Biomechanical Phenomena , Body Weight/drug effects , Dependovirus/drug effects , Dependovirus/metabolism , Dietary Supplements , Gene Transfer Techniques , Mice , Mice, Inbred mdx , Muscle Contraction , Muscle Fatigue , Muscle Fibers, Fast-Twitch/drug effects , Muscle Fibers, Slow-Twitch/drug effects , Muscular Dystrophy, Animal/complications , Myosins/metabolism , Organ Size , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Recovery of Function/drug effects , Resveratrol , Stilbenes/administration & dosage , Stilbenes/pharmacology , Transcription FactorsABSTRACT
PURPOSE: With the increasing number of retinal gene-based therapies and therapeutic constructs, in vitro bioassays characterizing vector transduction efficiency and quality are becoming increasingly important. Currently, in vitro assays quantifying vector transduction efficiency are performed predominantly for non-ocular tissues. A human retinal pigment epithelial cell line (ARPE19) and a mouse cone photoreceptor cell line, 661W, have been well characterized and are used for many retinal metabolism and biologic pathway studies. The purpose of this study is to quantify transduction efficiencies of a variety of self-complementary (sc) adeno-associated virus (AAV) vectors in these biologically relevant ocular cell lines using high-throughput fluorescence-activated cell sorting (FACS) analysis. METHODS: ARPE19 and 661W cells were infected with sc-smCBA-mCherry packaged in unmodified AAV capsids or capsids containing single/multiple tyrosine-phenylalanine (Y-F) mutations at multiplicity of infections (MOIs) ranging from 100 to 10,000. Three days post infection fluorescent images verified mCherry expression. Following microscopy, FACS analysis was performed to quantify the number of positive cells and the mean intensity of mCherry fluorescence, the product of which is reported as transduction efficiency for each vector. The scAAV vectors containing cone-specific (sc-mCARpro-green fluorescent protein [GFP]), rod-specific (sc-MOPS500-eGFP), retinal pigment epithelium (RPE)-specific (sc-VMD2-GFP), or ubiquitous (sc-smCBA-GFP) promoters were used to infect both cell lines at an MOI of 10,000. Three days post infection, cells were immunostained with an antibody raised against GFP and imaged. Finally, based on our in vitro results, we tested a prediction of transduction efficiency in vivo. RESULTS: Expression from unmodified scAAV1, scAAV2, scAAV5, and scAAV8 vectors was detectable by FACS in both ARPE19 and 661W cells, with scAAV1 and scAAV2 being the most efficient in both cell lines. scAAV5 showed moderate efficiency in both ARPE19 and 661W cells. scAAV8 was moderately efficient in 661W cells and was by comparison less so in ARPE19 cells; however, transduction was still apparent. scAAV9 performed poorly in both cell types. With some exceptions, the Y-F capsid mutations generally increased the efficiency of scAAV vector transduction, with the increasing number of mutated residues improving efficiency. Results for single scAAV1 and scAAV8 capsid mutants were mixed. In some cases, efficiency improved; in others, it was unchanged or marginally reduced. Retinal-specific promoters were also active in both cell lines, with the 661W cells showing a pattern consistent with the in vivo activity of the respective promoters tested. The prediction based on in vitro data that AAV2 sextuple Y-F mutants would show higher transduction efficiency in RPE relative to AAV2 triple Y-F capsid mutants was validated by evaluating the transduction characteristics of the two mutant vectors in mouse retina. CONCLUSIONS: Our results suggest that this rapid and quantifiable cell-based assay using two biologically relevant ocular cell lines will prove useful in screening and optimizing AAV vectors for application in retina-targeted gene therapies.
Subject(s)
Dependovirus/metabolism , Epithelial Cells/metabolism , Green Fluorescent Proteins/metabolism , Photoreceptor Cells/metabolism , Recombinant Fusion Proteins/metabolism , Retina/metabolism , Animals , Capsid/chemistry , Capsid/metabolism , Cell Line , Dependovirus/genetics , Epithelial Cells/cytology , Flow Cytometry , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors/chemistry , Green Fluorescent Proteins/genetics , High-Throughput Screening Assays , Humans , Mice , Phenylalanine/genetics , Phenylalanine/metabolism , Photoreceptor Cells/cytology , Recombinant Fusion Proteins/genetics , Retina/cytology , Transformation, Genetic , Transgenes , Tyrosine/genetics , Tyrosine/metabolism , Vision, OcularABSTRACT
Apolipoprotein A-I (ApoA-I)/high-density lipoprotein (HDL)-raising treatments are effective antiatherosclerotic strategies. We have compared the antiatherogenic effects of human ApoA-I (hApoA-I) overexpression by intraportal and intramuscular gene transfer in atherosclerotic ApoE-knockout mice. Atherosclerotic lesions were induced by atherogenic diet. After atherosclerosis induction, a group of animals was killed and served as atherosclerosis baseline-control group. The remaining animals were randomized into the following groups: (1) atherosclerosis-progression-control, (2) intraportal/vector administration, and (3) intramuscular/vector administration. Aortas and hearts were processed for atherosclerotic quantification by en face Sudan IV and Oil Red-O, respectively. Liver and muscle specimens were processed for protein/gene expression analysis. A sustained increase in hApoA-I/HDL plasma levels was observed in both transduced groups. hApoA-I overexpression abolished plaque progression versus progression-control group. hApoA-I overexpression significantly reduced lesion macrophage, feature indicative of plaque stabilization. Scavenger receptor class-B type I (SR-BI), but not ATP-binding cassette, sub-family A (ABCA), member 1 (ABCA-1), was significantly upregulated in treated groups versus progression-controls. The results of this study show a similar effect of hApoA-I/HDL overexpression on plaque progression/stabilization by 2 different routes of administration. Our results showing similar effects using either intramuscular administration and intraportal route of administration may have significant clinical implications, given the reduced medical risk to patient and cost of intramuscular injections.
Subject(s)
Aorta/drug effects , Apolipoprotein A-I/genetics , Apolipoprotein A-I/therapeutic use , Apolipoproteins E/genetics , Atherosclerosis/drug therapy , Dependovirus/metabolism , Liver/drug effects , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/analysis , ATP-Binding Cassette Transporters/genetics , Animals , Aorta/pathology , Apolipoprotein A-I/administration & dosage , Apolipoprotein A-I/blood , Apolipoproteins E/metabolism , Atherosclerosis/genetics , Atherosclerosis/metabolism , Atherosclerosis/pathology , Cholesterol, HDL/blood , Dependovirus/genetics , Diet, Atherogenic , Disease Progression , Drug Evaluation, Preclinical , Genetic Vectors , Humans , Injections, Intramuscular , Injections, Intravenous , Liver/anatomy & histology , Liver/physiopathology , Mice , Mice, Knockout , Molecular Targeted Therapy , Scavenger Receptors, Class B/analysis , Scavenger Receptors, Class B/genetics , Time Factors , Transduction, GeneticABSTRACT
BACKGROUND: This study compared the transduction efficiencies of an adeno-associated viral (AAV) vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP), with a lentiviral (LV) vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed), to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 x 108 or 1 x 109 genomic copies of AAV1-GFP and 1 x 105 transducing units of LV-dsRed. RESULTS: Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. CONCLUSIONS: This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.